Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the invention relates to methods of determining a predisposition for QT interval prolongation in a subject after the administration of a pharmaceutical agent or agents.
• Compositions and kits for determining said predispositions to the QT interval prolongation are also described.
• the invention relates to a method of screening a subject for a predisposition to an adverse drug reaction involving prolonged QT intervals.
• the genetic screening of patients for said predisposition focuses on genes associated with QT interval prolongation, including LQT genes, P-glycoprotein membrane pump proteins (P-gp), multidrug resistance genes and cytochrome P450-mediated drug metabolism genes.
• LQT long QT syndrome
• LQT4 KVLQT1
• HERG LQTl
• SCN5A LQT3
• MinK LQTS
• LQT6 a sixth gene (LQT6) has been identified (Wang et al., Ann. Med. 30: 58-65 (1998)). All but LQT3 encode cardiac potassium ion (K ) channel proteins; LQTS encodes a cardiac sodium ion (Na ) channel protein (Vincent, Annu. Rev. Med. 49: 263-74 (1998)).
• TdP Torsades de Pointes
• U.S. Patent No. 5,599,673 claims two (e.g., HERG and SCN5.4) of the six LQT genes. Two HERG-related genes have also been claimed (U.S. Patent No. 5,986,081).
• International PCT Application WO 97/23598 describes a method of assessing a patient's risk for long QT syndrome (LQTS) by screening for genetic mutations in the MinK gene.
• LQTS long QT syndrome
• these patents do not disclose methods of diagnosing a patient's predisposition to an adverse drug reaction involving elongation of the QT interval due to mutations in any of the LQT genes.
• Drugs have been identified that cause QT interval prolongation, arid thereby adverse drug reactions.
• Certain antihistamines such as terfenadine (e.g., Seldane®) and astemizole (e.g., Hismanal®), reportedly block potassium channels (Woosley, Annu. Rev. Pharmacol. Toxicol 36: 233-52 (1996)) and inhibit the H ⁇ RG protein, and thereby were postulated to induce Torsades de Pointes (Wang et al, 1998). All antiarrhythmic drugs that lengthen repolarization reportedly can cause Torsades de Pointes (Drici et al, Circulation 94: 1471-4 (1996)).
• cytochrome P450 enzymes have also been linked to adverse drug reactions.
• CYP2D6 was the first cytochrome P450 isoform found to be genetically polymorphic in its distribution (Eichelbaum et al, Eur. J. Clin. Pharmacol. 16: 183-7 (1979); and Mahgoub et al, Lancet 2: 584-6 (1977)), and it is now clear that -this enzyme metabolizes a large number of drugs (Inaba et al, Can. J. Physiol
• cytochrome P450 genes which are involved in the metabolism of drugs and drag metabolites. Several of them include CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP3A7. Allelic variations exist amongst these genes. Certain of these allelic variations combine to produce a poor metabolizer phenotype in 7% of Caucasians, but smaller percentages of Af icans and Asians and the "ultrarapid" phenotype in ⁇ 5% of Caucasian and up to 30% Africans. As ethnic-specific alleles for both Asians (Yokoi et al, Pharm. Res.
• U.S. Patent No. 5,891,633 relates to a method of identifying mutations in the cytochrome P450 genes CYP2C9 and CYP2A6.
• Japanese Patent No. 8168400 provides a method of determining mutations in exons 6 and 7 of the CYP2C19 gene.
• Japanese Patent No. 10014585 describes primers and methods of detecting a mutation in exon 5 of CYP2C19, which is related to the abnormal metabolism of diazepam, imipramine, omeprazole and propranolol.
• U.S. Patent No. 5,912,120 claims a method of diagnosing a patient having a deficiency in S-mephenytoin 4'-hydroxylase activity by detecting polymorphisms at nucleotides 681 or 636.
• U.S. Patent 5,719,026 provides methods and primers for detecting a polymorphisms in CYP1A2 and assessing the changes in the drug activity of theophylline associated with those polymorphisms.
• Japanese Patent No. 10286090 reportedly describes methods and primers to detect mutations in CYP2E1. These mutations are reported as being useful for determining the safety margin for drag administration for the treatment or related diseases.
• P-Glycoprotein Pump in the development of drag-resistant tumor cells has been extensively studied (Lo et al, J. Clin. Pharmacol. 39: 995-1005 (1999)).
• P- gp is an ATP-dependent drug pump that extrudes a broad range of cytotoxic agents from the cells end is encoded by a gene called MDR-1, for multidrug resistance (Loo et al, Biochem. Cell Biol 11: 11-23 (1999); and Robert, Eur. J. Clin. Invest. 29: 536- 45 (1999)).
• MDR-1 multidrug resistance
• the human P-gp sequence has been described by Chen et al, Cell 47: 381-9 (1986) and has the GenBank Accession No. M14758.
• Multidrug resistance can be diagnosed in tumors using molecular biology techniques (e.g., gene expression at the mRNA level), by immunological techniques (e.g., quantification of the P-glycoprotein itself) or by functional approaches (e.g., measuring dye exclusion) (Robert, 1999).
• molecular biology techniques e.g., gene expression at the mRNA level
• immunological techniques e.g., quantification of the P-glycoprotein itself
• functional approaches e.g., measuring dye exclusion
• Drags have been developed which reverse or modulate MDR.
• PSC-833 is a non-immunosuppressive cyclosporin derivative that potently and specifically inhibits P-gp (Atadja t ⁇ /., Cancer Metastasis Rev. 17: 163-8 (1998)).
• compounds have been identified which increase or modulate the bioavailability of pharmaceutical compounds. See, e.g., U.S. Patent Nos. 6,004,927; 5,962,522; 5,916,566; 5,716,928; 5,665,386; and 5,567,592.
• P-gp activity has been altered by expression of antisense nucleotides specific to MDR-1 (U.S. Patent No. 6,001,991). Methods and assays have also been developed which assess whether multidrug resistance has been reversed (U.S. Patent No. 5,403,574).
• nucleic Acid Hybridization The capacity of a nucleic acid "probe” molecule to hybridize (i. e., base pair) to a complementary nucleic acid "target” molecule forms the cornerstone for a wide array of diagnostic and therapeutic procedures.
• Hybridization assays are extensively used in molecular biology and medicine. Methods of performing such hybridization reactions are disclosed by, for example, SAMBROOK ETAL., MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), HAYMES ETAL., NUCLEIC ACID HYBRIDIZATION: A PRACTICAL APPROACH (IRL Press, Washington, D.C. (1985)) and KELLER ETAL., DNA PROBES (2 nd Ed., Stockton Press, New York (1993)).
• Single nucleotide polymorphisms differ significantly from the variable nucleotide type polymorphisms (VNTRs), that arise from spontaneous tandem duplications of di- or tri-nucleotide repeated motifs of nucleotides (Weber, U.S. Pat. No. 5,075,217; Armour et al, FEBS Lett. 307: 113-5 (1992); Horn et al, PCT Application No. WO 91/14003; Moore et al, Genomics 10: 654-60 (1991); Hillel et al, Genet.
• VNTRs variable nucleotide type polymorphisms
• restriction fragment length polymorphisms that comprise variations which alter the lengths of the fragments that are generated by restriction endonuclease cleavage (e.g., Fischer et al, (PCT Application No. W0 90/13668); and Uhlen (PCT Application No. WO 90/11369)).
• SNPs constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation; it is unnecessary to determine a complete gene sequence for each patient.
• Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.
• Cohen et al (French Patent 2,650,840; and PCT Application No. WO 91/02087) discuss a solution-based method for determining the identity of the nucleotide of a polymorphic site.
• a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site.
• Additional SNP detection methods include the Genetic Bit Analysis method described by Goelet et al (PCT Application No. 92/15712). The method of Goelet et al uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymorphic site. Cheesman (U.S. Pat. No.
• Gentalen et al, Nucl. Acids Res. 27: 1485-91 (1999) describe a cooperative hybridization method to establish physical linkage between two loci on a DNA strand by using hybridization to a new type of high-density oligonucleotide array. This same method can be used to determine SNP haplotypes.
• Yershov et al, Proc. Natl Acad. Sci. USA 93: 4913-8 (1996) describe an oligonucleotide microchip which has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. This technology was suggested for large scale diagnostics in gene polymorphism studies. Guo et al, Nucl. Acids Res.
• the methods comprise the step of screening a biological sample from the subject through a nucleic acid array, wherein said nucleic acid array contains probes for at least two genetic mutations or polymorphisms. These two genetic mutations or polymorphisms are located in two or more of the group of genes consisting of (1) LQT genes, (2) altered sensitivity genes, and (3) increased exposure genes.
• Preferred genes include LQT genes and MDR genes (e.g., MDR-1).
• the nucleic acid array can be in the form of a chip, a microchip, a bead or a microsphere.
• the LQT gene which may contain a polymorphism which induces QT interval elongation include LQTl, LQT2, LQT3, LQT4, LQT5 and LQT6.
• the method may further comprise screening both LQT and increased exposure gene (e.g., cytochrome P450 genes) mutations and polymorphisms.
• the P450 cytochrome isoforms which may contain a mutation which can result in excessive accumulation of drags and thereby induce QT interval elongation include: CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP2E1, CYP3A, CYP3A5 and CYP3A7 '.
• a further object of the invention is to provide a method for determining whether a subject has a predisposition for QT interval elongation (e.g., acquired
• LQTS when treated with one or more pharmaceutical agents
• a nucleic acid array such as a DNA array.
• the DNA array contains probes for two or more genetic mutations or polymorphisms in at least two or more groups of genes wherein the genes are selected from the group consisting of (1) LQT genes, (2) altered sensitivity genes (e.g., MiRP- 1 genes and its related genes), and (3) increased exposure genes (e.g., multidrug resistant genes and cytochrome P450 genes).
• the two or more genetic mutations or polymorphisms are found in these genes as at least one or more genetic mutations or polymorphisms in each of the two or more groups of genes.
• the genes can be selected from those described above.
• Another object of the invention is to provide a nucleic acid array comprising nucleic acids which recognize and bind to mutations of the QT syndrome genes (e.g., LQT genes), the altered sensitivity genes (MirR-1 genes) and/or the increased exposure genes.
• QT syndrome genes e.g., LQT genes
• MirR-1 genes the altered sensitivity genes
• Another object of the invention is to provide a method of screening one or more pharmaceutical agents in vitro for its or their ability to induce prolonged cardiac repolarization of a cell comprising the steps of A) measuring I ⁇ r and I RS currents of the cell using a voltage clamp before superfusing the cell with a candidate agent or agents; B) superfusing and incubating the cell with the candidate agent or agents; C) measuring the I r and I S currents after superfusion and incubation of the cell with the candidate agent or agents using a voltage clamp; and D) determining whether the I r and I S currents are inhibited or abolished thereby indicating that the drag prolongs repolarization.
• It is another object of the invention to provide a method for identifying genetic polymorphisms and mutations, which can cause QT interval prolongation in a subject comprising the steps of inserting at least two nucleic acids each encoding a polymorphism or mutation of at least two of the following genes: a LQT gene, an altered sensitivity gene, and/or an increased drug exposure gene into a cell; B) measuring I ⁇ r and I KS currents of the cell before administering a drug known to cause a change in I& and/or I ⁇ ; C) measuring fe and I KS currents of the cell after superfusion of the cell with the drag; D) comparing the Ij - and I KS values of the cell expressing the polymorphisms and or mutations to the l ⁇ r and I KS values of a cell expressing a wild- type genes; and E) determining if the presence of the polymorphisms and/or mutations leads to greater inhibition or blockage of I ⁇ r and I KS currents in
• FIG. 1 Recordings of I ⁇ » I to (A and B) and I ⁇ (C) in the same cell before and after 5 minutes superfusion with 5 ⁇ moI/L E-4031.
• Panel A I& and I 0 currents before and after superfusion with E-4031.
• E-4031 abolished the I & tail current and also reduced the time-dependent I ⁇ r current without affecting the transient outward current (I t0 ) or the holding current;
• Panel B E-4031 sensitive currents obtained by digital subtraction of currents after E-4031 exposure from currents before E-4031 exposure. Note the inward rectification of the time-dependent I ⁇ currents at very positive potentials compared with the tail currents;
• Panel C I ⁇ current before and after superfusion with E-4031.
• E-4031 showed little effect on the inward I- ⁇ current recorded at -120 mV. The outward holding currents that represent the amplitude of I ⁇ at -40 mV are superimposed.
• FIG. 1 Recordings of I ⁇ o I o (A and B) and I ⁇ (C) in the same cell before and after 5 minutes superfusion with 10 ⁇ mol/L tamoxifen.
• Panel A) !& and I t0 currents recorded before and after superfusion with tamoxifen. Tamoxifen abolished the I ⁇ r tail current and also reduced the time-dependent I r current, without affecting the transient outward current (I t0 );
• Panel B Tamoxifen-sensitive currents obtained by digital subtraction of currents before and after tamoxifen superfusion.
• I ⁇ r currents recorded from the same cell before drug administration 5 minutes after superfusion with 10 ⁇ mol/L tamoxifen and 5 minutes after washout. I ⁇ r tail currents were abolished by tamoxifen without recovery.
• Panel C) Inhibition of I ⁇ r by 3.3 ⁇ mol/L tamoxifen and 3.3 ⁇ mol/L quinidine. Data are expressed as mean ⁇ SD, n 4, **p ⁇ 0.01.
• Figure 6 Effect of tamoxifen on the action potential duration (APD). Action potentials were elicited by injecting 100 pA depolarizing currents of 2 ms, at a frequency of 0.45 HZ. Shown in the figure are two superimposed action potential tracings recorded in a single ventricular myocyte before and 4 minutes after exposure to 3.3 ⁇ mol/L tamoxifen. The two tracings are averaged tracings from 16 trials.
• FIG. 7 Effect of tamoxifen on the L-type Ic a - lea was recorded in the same cell before tamoxifen administration and 1, 2, 3, 4 min. after superfusion of 10 ⁇ mol/L tamoxifen and after 2, 4, 8 and 16 min. after washout. Note the marked inhibition of Ic a and partial recovery after washout.
• the invention involves a method for diagnosing a subject's predisposition to an adverse drug response involving a prolonged QT interval, resulting from an excessive accumulation of drags due to genetic polymorphisms or mutations in at least two classes of genes, which can result in potentially fatal cardiac arrhythmia.
• the drags cover an array of pharmaceuticals including anti-arrhythmics, anti- psychotics, antidepressants, anti-anginals, antibiotics, anti-fungals, anti-virals, diuretics, migraine drags, mental illness therapeutics, breast cancer therapeutics, anxiolytics, anti-nausea agents, cardiac medication, opiate agonists, antihypertensives, antiinfectives, and anticonvulsants.
• the inventive method for determining the adverse drag reaction potential utilizes a DNA array (e.g., DNA chip), which can be used to assay a biological sample from a patient.
• a DNA array e.g., DNA chip
• a patient's DNA sample could be run through a DNA array to diagnose whether the patient has any genetic mutations or polymorphisms that are associated with prolonging cardiac repolarization.
• Preferred genes, which are associated directly or indirectly with prolonging repolarization include LQT genes, altered sensitivity genes (e.g., MiRPl genes or related genes), and increased exposure genes (cytochrome P450 genes and MDR genes).
• bp or “base pair” is meant the hydrogen bonded purine and pyrimidine pair in a double-stranded nucleic acid.
• the pairs are adenine (A) and thymine (T), and guanine (G) and cytosine (C).
• the pairs are adenine (A) and uracil (U). and guanine (G) and cytosine (C).
• nucleotide or “nt” is meant the nucleotide, typically a deoxyribonucleic acid, of the type adenine (A), thymine (T), guanine (G), uracil (U), and cytosine (C) typically in the sense or coding orientation, but can also include antisense orientations of the gene or coding sequence.
• A adenine
• T thymine
• G guanine
• U uracil
• C cytosine typically in the sense or coding orientation, but can also include antisense orientations of the gene or coding sequence.
• nucleic acid or “nucleic acid molecule” is meant a deoxyribonucleotide or ribonucleoti.de polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
• aa is meant amino acid.
• genes are meant a unit of inheritance that occupies a specific locus on a chromosome (chr.), the existence of which can be confirmed by the occurrence of different allelic forms.
• Preferred genes of this application are those which impact cardiac repolarization, especially those which act to prolong cardiac repolarization, genes which determine the elimination of drugs from a host, and genes which prolong the time necessary to eliminate a drug from the host. This can include cytochrome P450 genes and ion channel genes.
• mutation is meant a one or more nucleotide change in the DNA or RNA sequence of an organism.
• mutation can be a frame-shift mutation, a nonsense mutation, or a missense mutation.
• polymorphism is meant the existing, in a population, of two or more alleles of a gene, wherein the frequency of the rarer alleles is greater than can be explained by recurrent mutation alone (typically greater than one percent). Said polymorphisms can consist of one or more nucleotide differences. The polymorphisms can be silent, wherein they do not confer a change in the associated amino acid sequence. Alternatively, the polymorphism can cause an associated change in the amino acid sequence encoded by the gene.
• altered sensitivity genes is meant to include genes, which when mutated, alter the expression of proteins which thereupon results in altered sensitivity to a drag or to drugs.
• genes can include for example, the potassium ion channel gene, MiRPl or related genes.
• increased exposure gene is meant to include genes which when aberrantly expressed in a subject lead to increased exposure to a drag or drags.
• genes can include cytochrome P450 genes, which when mutated lead to decreased or aberrant expression of enzymes required for the elimination of drags or of a drug.
• Increased exposure genes also include multidrug resistance (MDR) genes. Mutations in MDR genes can lead to altered distribution (and thereby increased exposure) of a drug or drugs in tissues or in a tissue.
• MDR genes encode membrane drug transporters and/or ion channel proteins.
• ion channel gene is meant to include multidrug resistence genes (MDR genes) as well as ion pump genes such as the LQT family of genes, certain sodium (Na “1” ) chamiel genes (see, e.g., Chen et al, Nature 392: 293-6 (1998)) and certain potassium (K*) channel genes.
• MDR genes multidrug resistence genes
• ion pump genes such as the LQT family of genes, certain sodium (Na “1” ) chamiel genes (see, e.g., Chen et al, Nature 392: 293-6 (1998)) and certain potassium (K*) channel genes.
• the potassium ion channel gene, MiRPl is one preferred example of a potassium ion channel which may be linked to QT interval prolongation; MiRPl protein forms channels with HERG and its mutations are associated with cardiac arrhythmia.
• Preferred MDR genes include MDR-1 which encodes P-glycoprotein pump (P-gp).
• Prolonged QT interval By “prolonged QT interval,” “QT interval prolongation” or “QT interval elongation” is meant the QT interval measured from QRS onset to T wave offset (QTo) and from QRS onset to T wave peak (QTm) adjusted to a heart rate of 60 beats per minute, which is QTc.
• QTc By “QTc” is also referred to as the Bazett corrected QT interval. See, e.g., Kligfield et al, J. Am. Coll. Cardiol 28: 1547-55 (1996).
• Prolonged QT intervals can be induced directly or indirectly by at least two genetic mutations or polymorphism. These mutations or polymorphisms are located in two or more groups of genes (e.g., at least one mutation or polymorphism per gene group), wherein the groups are (1) LQT genes, (2) altered sensitivity genes (e.g., MiRPl genes), or (3) increased exposure genes (e.g., MDR genes or cytochrome P450 genes).
• TdP is an uncommon variant of ventricular tachycardia (VT). The underlying etiology and management of TdP are, in general, quite different from the more common ventricular tachycardia.
• TdP is a polymorphous ventricular tachycardia in which the morphology of the QRS complexes vary from beat to beat.
• the ventricular rate can range from about 150/min to about 250/min. hi most cases, there is a constantly changing wave form, but there may not be regularity to the axis changes.
• the definition also requires that the Q-T interval be markedly increased (usually to 600 msec or greater). Cases of polymorphic VT, which are not associated with a prolonged Q-T interval, are treated as generic VT.
• TdP usually occurs in bursts that are not sustained, thus, one usually has a rhythm strip showing the patient's base-line Q-T prolongation.
• predisposition is meant a tendency for a subject to develop TdP de Pointes or QT interval elongation. This tendency may be acquired or hereditary.
• the preferred subject is a human subject.
• the predisposition is related to induction of TdP or QT interval elongation upon the administration of one or more pharmaceutical agents which induce TdP or QT interval elongation. These pharmaceutical agents can be those listed herein or any later identified investigational drag which induces QT interval prolongation.
• LQTS or “long QT syndrome” is meant a genetic disease which predisposes individuals to ventricular arrhythmias that lead to syncope and sudden death.
• Congenital or idiopathic LQTS is an inherited form of the disease and is genetically heterogeneous (Wei et al, Circulation 92: 1-275 (1995)) and includes the Jervell-Lange-Nielsen and the Romano-Ward-syndromes (Napolitano et al, Drugs 47: 51-65 (1994)).
• Acquired prolonged QT syndromes are largely iatrogenic, and may be induced by certain drugs or associated with metabolic disturbances (e.g., hypokalemia, hypocalceniia or hypomagnesemia) (Napolitano et al, 1994).
• nucleic acid array is meant a substrate to which one oir more, preferably
• arrays with 5,000 to 500,000 nucleic acids attached.
• a DNA chip array For example, see U.S. Patent Nos. 5,981,956 and 5,922,591.
• Other examples include Gene Logic's Flow-thra ChipO Probe ArraysO (U.S. Patent No. 5,994,068) or the FlowMetrix technology (e.g., microspheres) of Luminex. These arrays are contemplated to contain nucleic acids for wild-type and mutated genes encoding ion channel genes and/or cytochrome P450 genes or isoforms thereof.
• mutation By “mutation,” “mutant” or “mutated” is meant to refer to a genetic change (e.g., frame-shift mutation, non-sense mutation, missense mutation, deletion, or insertion) in a gene (e.g., ion channel gene, P-gp or a cytochrome P450 gene) resulting in an altered gene expression and/or altered protein function.
• a genetic change e.g., frame-shift mutation, non-sense mutation, missense mutation, deletion, or insertion
• a gene e.g., ion channel gene, P-gp or a cytochrome P450 gene
• isoform is meant different forms of a protein that may be produced from different genes or from the same gene by alternative RNA splicing.
• binds substantially is meant to complementary hybridization between an oligonucleotide and a target sequence.
• hybridizing is meant the binding of two single stranded nucleic acids via complementary base pairing.
• primer refers to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
• a primer is preferably a single-stranded oligodeoxyribo-nucleotide. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides.
• Primer generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
• a primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.
• the term "primer” may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding one or both ends of the target region to be amplified. For instance, if a region shows significant levels of polymorphism or mutation in a population, mixtures of primers can be prepared that will amplify alternate sequences.
• a primer can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
• useful labels include P, fluorescent dyes, electron-dense reagents, enzymes (as commonly used in an ELISA), biotin, or haptens and proteins for which antisera or monoclonal antibodies are available.
• a label can also be used to "capture" the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support.
• pharmaceutical agent refers to an agent or drag which alone or in combination with one or more other pharmaceutical agents can induce in a patient prolonged cardiac repolarization. The specific pharmaceutical agents which may induce QT interval elongation are provided herein.
• I r is meant the major rapid repolarizing current in a cell also known as rapid component of the delayed rectifier potassium current.
• 1- ⁇ s is meant the slower component of the delayed rectifier current.
• I ⁇ is meant inward rectifier current. Both I ⁇ r and Ija are forms of potassium current densities.
• I to is meant the transient outward current of a cell as measured in a voltage clamp assay.
• biological sample or “sample” is meant a collection of biological material from a subject containing nucleated cells.
• This biological material may be solid tissue, for example from a fresh or preserved organ or tissue sample, biopsy or buccal swab; blood or blood constituents; bodily fluids such as amniotic fluid, peritoneal fluid, or interstitial fluid, etc.
• the sample may contain compounds which are not naturally intermixed with the biological material such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.
• One embodiment of the invention is to identify a pharmaceutical agent or combination of agents that induces QT interval elongation in a subject, especially a human.
• Agents or combinations of agents that induce elongation of the QT interval include, but are not limited to, those listed in Table 2, below. Several of these drags have been analyzed and have been identified which prolong the QT interval in a concentration-dependent manner (see, e.g., Drici et al, J. Clin. Psychopharmacol. 18: 477-81 (1998)). Table 2 Pharmaceutical Agents
• Nucleic acids which recognize these mutations can be placed in an array on a subsfrate, such as on a chip (e.g., DNA chip or microchips). These arrays also can be placed on other substrates, such as microtiter plates, beads or microspheres. Methods of linking nucleic acids to suitable substrates and the substrates themselves are described, for example, in U.S. Patent Nos. 5,981,956; 5,922,591; 5,994,068 (Gene Logic's Flow-thru ChipO Probe ArraysO); 5,858,659; 5,753,439; 5,837,860 and the FlowMetrix technology (e.g., microspheres) of Luminex (U.S. Patent Nos.
• the nucleic acids that recognize the polymorphisms of the LQT and cytochrome P450 genes preferably can be linked to a single substrate.
• the substrate may only comprise a single or a few (e.g., 2, 3, 4, 5, or 10) nucleic acids and may be mixed with microspheres comprising different nucleic acids.
• a second, more preferred method for nucleic acid array synthesis utilizes an automated DNA synthesizer for DNA synthesis.
• the controlled chemistry of an automated DNA synthesizer allows for the synthesis of longer, higher quality DNA molecules than is possible with the first method.
• the nucleic acid molecules synthesized can be purified prior to the coupling step.
• the nucleic acids can be attached to the substrate as described in U.S. 5,837,860.
• covalently immobilized nucleic acid molecules may be used to detect specific PCR products by hybridization where the capture probe is immobilized on the solid phase or subsfrate (Ranki et al, Gene 21 : 77-85 (1983);
• a preferred method would be to prepare a single-stranded PCR product before hybridization.
• a patient sample that is suspected to contain the target molecule, or an amplification product thereof, would then be exposed to the solid-surface and permitted to hybridize to the bound oligonucleotide.
• the methods of the present invention do not require that the target nucleic acid contain only one of its natural two sfrands.
• the methods of the present invention may be practiced on either double-stranded DNA (dsDNA), or on single-stranded DNA (ssDNA) obtained by, for example, alkali treatment of native DNA.
• dsDNA double-stranded DNA
• ssDNA single-stranded DNA obtained by, for example, alkali treatment of native DNA.
• alkali treatment of native DNA for example, alkali treatment of native DNA.
• the presence of the unused (non-template) strand does not affect the reaction.
• any of a variety of methods can be used to eliminate one of the two natural stands of the target DNA molecule from the reaction.
• Single-stranded DNA molecules may be produced using the ssDNA bacteriophage, M13 (Messing et al, Meth. Enzymol. 101: 20-78 (1983); see also, SAMBROOK ETAL.,
• Screening Polymorphisms Screening polymorphisms in samples of genomic material according to the methods of the present invention, is generally carried out using arrays of oligonucleotide probes. These arrays may generally be “tiled” for a large number of specific polymorphisms.
• tileing is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basic set of monomers, i.e. nucleotides. Tiling strategies are discussed in detail in Published PCT Application No.
• target sequence is meant a sequence which has been identified as containing a polymo ⁇ hism or mutation (e.g., a single-base polymorphism also referred to as a "biallelic base”). It will be understood that the term “target sequence” is intended to encompass the various forms present in a particular sample of genomic material, i.e., both alleles in a diploid genome.
• arrays are tiled for a number of specific, identified polymorphic marker sequences.
• the array is tiled to include a number of detection blocks, each detection block being specific for a specific polymorphic marker or set of polymorphic markers.
• a detection block may be tiled to include a number of probes which span the sequence segment that includes a specific polymorphism.
• the probes are synthesized in pairs differing, for example, at the biallelic base. Ih addition to the probes differing at the biallelic bases, monosubstituted probes can be generally tiled within the detection block.
• These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C or U).
• the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the base that corresponds to the polymorphism.
• bases up to and including those in positions 2 bases from the polymorphism will be substituted.
• the monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artifactual cross-hybridization.
• a detection block may be tiled to provide probes having optimal hybridization intensities with minimal cross-hybridization.
• a sequence downstream from a polymo ⁇ hic base is G-C rich, it could potentially give rise to a higher level of cross-hybridization or "noise," when analyzed. Accordingly, one can tile the detection block to take advantage of more of the upstream sequence.
• Optimal tiling configurations may be determined for any particular polymo ⁇ hism by comparative analysis. For example, triplet or larger detection blocks may be readily employed to select such optimal tiling strategies.
• arrays will generally be tiled to provide for ease of reading and analysis.
• the probes tiled within a detection block will generally be arranged so that reading across a detection block the probes are tiled in succession, i.e., progressing along the target sequence one or more nucleotides at a time.
• the target nucleic acid is hybridized with the array and scanned.
• Hybridization and scanning are generally carried out by methods described in, e.g., PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Patent No.
• a target nucleic acid sequence which includes one or more previously identified polymo ⁇ hic markers, is amplified by well known amplification techniques, e.g., polymerase chain reaction (PCR). Typically, this involves the use of primer sequences that are complementary to the two sfrands of the target sequence both upstream and downstream from the polymo ⁇ hism. Asymmetric PCR techniques may also be used.
• Amplified target generally inco ⁇ orating a label, is then hybridized with the array under appropriate conditions. Upon completion of hybridization and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes.
• the hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array.
• the arrays of the invention will include multiple detection blocks, and thus be capable of analyzing multiple, specific polymo ⁇ hisms.
• preferred arrays will generally include from about 50 to about 4,000 different detection blocks with particularly preferred arrays including from 10 to 3,000 different detection blocks.
• detection blocks may be grouped within a single array or in multiple, separate arrays so that varying, optimal conditions may be used during the hybridization of the target to the array. For example, it may often be desirable to provide for the detection of those polymo ⁇ hisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. Tins allows for the separate optimization of hybridization conditions for each situation.
• Additional methods of detecting gene mutations includes the methods described in International PCT applications WO 99/42622; WO 99/29901 ; WO 98/49341 ; WO 97/27317; and WO 97/22720.
• the hybridization data from the scanned array is then analyzed to identify which variant or variants of the polymo ⁇ hic marker are present in the sample, or target sequence, as determined from the probes to which the target hybridized, e.g., one of the two homozygote forms or the heterozygote form. This determination is termed "calling" the genotype. Calling the genotype is typically a matter of comparing the hybridization data for each potential variant, and based upon that comparison, identifying the actual variant (for homozygotes) or variants (for heterozygotes) that are present.
• this comparison involves taking the ratio of hybridization intensities (corrected for average background levels) for the expected perfectly hybridizing probes for a first variant versus that of the second variant.
• this ratio will be a large number, theoretically approaching an infinite value.
• homozygous for the second variant the ratio will be a very low number, i.e., theoretically approaching zero.
• the ratio will be approximately 1. These numbers are, as described, theoretical.
• the first ratio will be well in excess of 1, i.e., 2, 4, 5 or greater.
• the second ratio will typically be substantially less than 1, i.e., 0.5, 0.2, 0.1 or less.
• the ratio for heterozygotes will typically be approximately equal to 1, i.e., from 0.7 to 1.5. These ratios can vary based upon the specific sequence surrounding the polymo ⁇ hism, and can also be adjusted based upon a standard hybridization with a control sample containing the variants of the polymo ⁇ hism.
• the quality of a given call for a particular genotype may also be checked.
• the maximum perfect match intensity can be divided by a measure of the background noise (which may be represented by the standard deviation of the mismatched intensities). Where the ratio exceeds some preselected cut-off point, the call is determined to be good. For example, where the maximum intensity of the expected perfect matches exceeds twice the noise level, it might be termed a good call. Further description of software used for genetic calling can be used as described in U.S. Patent No. 5,858,659.
• Another aspect of the invention is to identify polymo ⁇ hisms or mutations which are associated with or indirectly involved with QT interval prolongation.
• These polymo ⁇ hisms or mutations are located in at least two classes of genes (e.g., LQT genes, altered sensitivity genes or increased exposure genes).
• the mutations or polymo ⁇ hisms can be those which have been previously identified but not linked with QT interval elongation.
• these also can be assessed using the assays described herein to determine whether the "new mutation” and/or "polymo ⁇ hism” can cause QT interval elongation.
• nucleic acids which recognize these polymo ⁇ hisms can be added to the nucleic acid array for screening subjects.
• One method of identifying such "new" polymo ⁇ hisms is to obtain biological samples from subjects who have experienced acquired LQTS due to administration of a drag or drugs and to sequence the LQT genes or P450 genes to isolate the polymo ⁇ hism which was responsible for the adverse drug reaction.
• the drugs can be assessed for their ability to elongate the QT interval as discussed.
• agents can be assayed using the Langendorff technique in, for example, isolated perfused rabbit hearts, or the whole-cell patch-clamp technique in ventricular myocytes to examine the TdP difference.
• Langendorff technique in, for example, isolated perfused rabbit hearts, or the whole-cell patch-clamp technique in ventricular myocytes to examine the TdP difference.
• Liu et al, J. Cardiovasc. Pharmacol. 34: 287-94 (1999) and Ebert et al, J. Womens Health 1: 547-57 (1998) used these techniques to examine the gender difference of Torsades de Pointes (TdP) between men and women.
• the whole-cell patch-clamp technique was utilized to study the effect of tamoxifen on the delayed rectifier (fc), the inward rectifier (I K ⁇ ), the transient outward current (I to ) and the inward L-type calcium current (Ic a ) in rabbit ventricular myocytes (Liu et al, J. Pharmacol Exp. Ther. 287: 877-83 (1998)).
• determination of the effect of a particular compound can also be performed by using an electrocardiogram (ECG) to study the pharmokinetics of a drug's effect on QTC prolongation (see, e.g., Sale et al, Clin. Pharmacol Ther. 56: 295-301 (1994)).
• ECG electrocardiogram
• the cells are fransfected with a nucleic acid with the putative mutation believed to cause QT prolongation when exposed to an agent known to cause QT elongation.
• the I r and I RS , and even the I to responses are measured and compared to a cell expressing the normal wild type gene. This can also be done when testing Na + current changes.
• the effect of the genetic mutations can also be assessed using voltage sensitive dye methods. Assaying changes to cell voltage using dyes are known in the art. See, for example, Morley et al, J. Cardiovasc. Eletrophysiol 10: 1361-75 (1999) and Dillon et al, Science 214: 453-6 (1981). The following examples are offered to illustrate embodiments of the invention, and should not be viewed as limiting the scope of the invention.
• Example 1 Method and kit for determining a subject's predisposition to adverse reactions to tamoxifen.
• the metabolic pathways of tamoxifen are complex and have been extensively studied. Tamoxifen metabolism involves multiple pathways, and the primary and secondary metabolites have variable pharmacological activities, with certain of the metabolites causing considerable inter-individual variability.
• the main routes of tamoxifen metabolism include N-demethylation, N-oxidation and 4-hydroxylation (Buckley et al, Drugs 37: 451-90 (1989); d Um et al, Carcinogenesis 15: 589-93 (1994)).
• Tamoxifen N- demethylation which appears quantitatively the most important pathway, is primarily catalyzed by CYP3A (Jacolot et al, Biochem. Pharmacol. 41 : 1911-9 (1991); (Mani et al, Drug Metab. Dispos. 21: 645-56 (1993)).
• CYP2D6 also appears to be a major enzyme that catalyzes tamoxifen 4-hydroxylation (Crewe et al, Biochem. Pharmacol. 53: 171-8 (1997)).
• Blood is drawn from a subject, e.g., human, into sodium heparin Vacutainers (Becton Dickinson; Franklin Lakes, NJ) and then is transferred to Corning 5.0 ml cryogenic vials (Corning; Cambridge, MA). The blood is frozen in a non-frost free -20°C freezer until use.
• Reagents which are used to extract the DNA from whole blood, are the QIAGEN Blood Midi Kit® unless otherwise noted.
• 200 ⁇ l of QIAGEN protease is added to a 15 ml centrifuge tube.
• the blood is thawed and 2.0 ml added to the tube followed by 2.0 ml of Buffer AL.
• the tube is capped and contents mixed with a vortexer for 15 seconds. After vortexing, 2 ml of 95% ethanol is added, and the contents mixed by inversion.
• the contents of the tube is then added to a QIAGEN Midi Spin column that is placed in a collection tube. The column and collection tube are centrifuged at 6,000 x g at 4°C for two minutes.
• the collection tube is discarded and replaced with a new collection tube.
• Two ml of Buffer AW is added to the spin column, and the column is centrifuged again at 6,000 x g at 4°C for two minutes.
• the column is rinsed again by discarding the collection tube, replacing it with a new collection tube, adding 2.0 ml of Buffer AW to the spin column, and centrifuging at 6,000 x g at 4°C for two minutes.
• the used collection tube is discarded and replaced with a new collection tube.
• the DNA is eluted from the column by adding 1 ml of elution buffer to the spin column, incubating at room temperature for one minute, and then centrifuging at 6,000 x g at 4°C for two minutes.
• the DNA is transferred to a Sarstedt 2.0 ml screw-top tube (#72730006, Sarstedt; Newton, NC) and frozen at -20°C.
• the DNA concentration is measured using the 260/280 method in a specfrophotometer, and the DNA concentration adjusted to 60 ng/ ⁇ l with water.
• DNA from buccal swabs is obtained as follows.
• Buccal cells are obtained by gently rabbing a sterile cotton swab within the subject's mouth.
• the swab is placed in a Falcon 2063 tube, and 1.5 mis of IX PBS is added, mixed, and centrifuged at 3K for 5 minutes to pellet the cells. The supernatant is removed, and this process is repeated with another 1 ml of IX PBS.
• the cell pellet is then suspended in 47 ⁇ l of PCR lysis buffer (Promega PCR Buffer B and 10 mg/ml Proteinase K). Samples are incubated for 30 minutes at 60°C, and the reaction stopped by boiling the sample for 10 minutes. The sample is then centrifuged at 3K for 5 minutes and stored at -20°C until use.
• PCR lysis buffer Promega PCR Buffer B and 10 mg/ml Proteinase K
• Reagents used in this protocol use the Affymetrix P450 GeneChip Kit® unless otherwise noted.
• the reaction mass mix is prepared in a template free area by combining the following:
• 96-well tube/tray retainer and the assembly is placed in a MicroAmp® base (all Perkin-Elmer; Foster City, CA). Forty-five ⁇ l of the mass mix is aliquoted into the 0.2 ml MicroAmp tubes and the tubes are capped. The tubes are then transferred to a medium template area to add the DNA.
• MicroAmp® base all Perkin-Elmer; Foster City, CA.
• the samples are then placed in a Perkin-Elmer GeneAmp® PCR System 9600 thermocycler programmed for: 95°C for 5 minutes, then 15 cycles of 95°C/40 seconds, 65°C/50 seconds, 72°C/50 seconds followed by 30 cycles of 95°C/30 seconds, 65°C/50 seconds, 72°C/50 seconds plus one second per cycle then a final extension of 72°C for 7 minutes.
• a Perkin-Elmer GeneAmp® PCR System 9600 thermocycler programmed for: 95°C for 5 minutes, then 15 cycles of 95°C/40 seconds, 65°C/50 seconds, 72°C/50 seconds followed by 30 cycles of 95°C/30 seconds, 65°C/50 seconds, 72°C/50 seconds plus one second per cycle then a final extension of 72°C for 7 minutes.
• a clean reaction tube rack is placed on ice with one tube for each sample that is to be fragmented. All fragmentation mass mix reagents are kept on ice, and the mass mix prepared by combining the reagents as directed in Table 6.
• the labeling master mix is prepared by combining the reagents in a microcentrifuge tube as follows:
• Hybridization Preparation The following solutions are prepared according to the direction in the Affymetrix® GeneChip® CYP450 instruction booklet: Hybridization Concentrate: 5.5 X SSPE, 0.055% Triton® X-100, and 1.1 mM CTAB
• the tubes are kept on ice at least 10 minutes. While the tubes are on ice, the Affymetrix® Fluidics Station is primed with water. Wash Buffer A (3X SSPE, 0.005% Triton X- 100 and 1 mM CTAB) and Wash Buffer B (6X SSPE).
• Wash Buffer A (3X SSPE, 0.005% Triton X- 100 and 1 mM CTAB) and Wash Buffer B (6X SSPE).
• a CYP450 Probe array is placed in the Fluidics Station module, and one of the Hybridization Master Mix/DNA tubes placed in the lower compartment. The CYP450 Hybridization protocol is run on the Fluidics Station.
• the hybridized chip is transferred from the Fluidics Station to the Scanner.
• a scan protocol is then ran on the chip as directed by
• Affymetrix® After the scan is completed, the Analysis program is run and the report prepared. The results are entered into an appropriate database.
• the tubes are briefly centrifuged in a microcentrifuge and placed in a Perkin Elmer® model 480 thermocycler.
• the amplification program used is 2 minutes at 94°C denaturation; 35 cycles of 1 minute at 94°C; 1 minute at 63°C; 1 minute at 72°C; with a final extension of 4 minutes at 72°C. Restriction Enzyme Digestion.
• Wild-type allele produces 110 and 182 base pair fragments.
• the 4 mutant produces a 292 base pair fragment and a heterozygote shows 110, 182 and 292 base pair bands on the gel.
• Conventional CYP2D6 * 10 Assay Amplification.
• the assay that is used for CYP2D6 * 10 is a two amplification allele specific oligonucleotide method.
• Amplification 1 Five ⁇ l of DNA (60 ng/ ⁇ l) is added to 45 ⁇ l PCR reaction mix containing IX PCR buffer B (Promega; Madison, Wl) [50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.0% Triton X-100]; 25 pmol of each primer, 200 ⁇ M of each dNTP, 1.0 mM MgCl 2 and 0.75 U of Taq polymerase (Promega, Madison, Wl) in 200 ⁇ l PCR tube strips.
• IX PCR buffer B Promega; Madison, Wl
• 50 mM KCl 10 mM Tris-HCl (pH 9.0), 1.0% Triton X-100]
• 25 pmol of each primer 200 ⁇ M of each dNTP, 1.0 mM MgCl 2 and 0.75 U of Taq polymerase (Promega, Madison, Wl) in 200 ⁇ l PCR tube strips.
• the primers used are: 5'-ACC AGG CCC CTC CAC CGG-3' upstream (primer 9) and 5'-TCT GGT AGG GGA GCC TCA GC-3' downstream (primer 10).
• the tube rack is briefly centrifuged in a table top centrifuge and placed in a Perkin Elmer® model 9600 thermocycler programmed to run 4 minutes at 94°C denaturation; 35 cycles of 1 minute at 94°C; 1 minute at 58°C, 1 minute at 72°C; with a final extension of 4 minutes at 72°C. program.
• Amplification 2 Two mass mixes are made for each sample.
• the wild-type mass mix contains primers 9 and 11 (primer 11 is 5'-CCA CCA GGC CCC CT-3') and the mutant mass mix contains primers 9 and 12 (5'-GCA CCA GGC CCC GT-3').
• the tube rack is briefly centrifuged in a tabletop centrifuge and placed in a Perkin Elmer® model 9600 thermocycler which is programmed to ran the 4 minutes at 94°C denaturation; 35 cycles of 1 minute at 94°C; 1 minute at 52°C; 1 minute at 72°C; with a final extension of 4 minutes at 72°C programmed.
• Gel Electrophoresis The samples are elecfrophoresed in pairs (wild type and mutant) on a 2.0%) agarose gel. Wild-type allele produces a 516 bp product only with the wild type reaction mix. The * 10 mutant allele produces a 516 bp product only with the mutant reaction mix. A heterozygote produces product with both mixes.
• the tubes are briefly centrifuged in a microcentrifuge and placed in a Perkin Elmer® model 480 thermocycler.
• the amplification program used is 2 minutes at 94°C. denaturation; 35 cycles of 1 minute at 94°C; 1 minute at 50°C; 1 minute at 72°C; with a final extension of 4 minutes at 72°C.
• Amplification Five ⁇ l of DNA (60 ng/ ⁇ l) is added to 20 ⁇ l of a PCR reaction mix containing IX PCR buffer B (Promega; Madison, Wl) [50 mM KCl, 10 mM Tris- HCl (pH 9.0), 1.0% Triton X-100]; 25 pmol of each primer, 200 ⁇ M of each dNTP, 1.5 mM MgCl 2 and 0.75 U of Taq polymerase (Promega; Madison, Wl) in 500 ⁇ l PCR reaction tubes. 25 ⁇ l of mineral oil is added to the top of each of the reaction tubes.
• the primers used are those of Bhasker et al.
• I ⁇ r is one of the major polarizing currents and its block has been implicated in TdP (Carlsson et al, J. Cardiovasc. Pharmacol. 16: 276-85 (1990); Roden et al, Am. Hear. J il l: 1088-93 (1986); Woosley, Annu. Rev. Pharmacol Toxicol 36: 233-52 (1996); and Follmer et al, Circulation 82: 289-93 (1990)).
• tamoxifen affects I ⁇ r we first establish the presence of I ⁇ r in the chosen model, rabbit ventricular myocytes, using a drag known to be specific for blocker of ! & .
• Figure 1 A and IB show the membrane currents elicited by a 1.5-second voltage-clamp step from -40 mV to different test potentials ranging from -10 to -50 mV in the same cell before (Panel A) and after (Panel B) 5 minutes exposure to 5 ⁇ mol/L E-4031, a highly selective I ⁇ r blocker (Clay et al, Biophys J. 69: 1830-7 (1995); and Sanguinetti et al, J. Gen. Physiol 961: 195-215 (1990)).
• FIG. 2B depicts the tamoxifen sensitive currents obtained by digital subtraction of currents in the bottom tracings from currents in the top tracings in panel A.
• the time-dependent current demonstrated strong inward rectification at very positive potentials compared with the tail current, while I t0 was not detectable in either the tamoxifen or the E-4031 sensitive current.
• Figure 2C shows the I ⁇ current measured before and after 5 minutes exposure to 10 ⁇ mol/L tamoxifen in the same cell shown in Figures 2A and 2B.
• tamoxifen produced no inhibition of the I ⁇ inward current at -120 mV.
• I ⁇ inward current was slightly larger after tamoxifen treatment of this cell (Fig. 2C).
• the outward holding currents representing the amplitude of I ⁇ at -40 mV were superimposable, indicating that tamoxifen had no effect on the I ⁇ outward current.
• Figure 3 depicts a typical experiment performed in the same cell before drag administration (control), or after 3, 5 and 9 min.
• I ⁇ r block by tamoxifen is time-dependent and has a slow onset. Further block can still be observed after superfusion for 5 minutes.
• I ⁇ r was readily recorded from control myocytes (no exposure to tamoxifen) for at least ten minutes without any sign of run-down.
• FIG. 5C compares the percentage inhibition of I ⁇ r by tamoxifen and quinidine at the same concentration of 3.3 ⁇ mol/L. These data show that, at the least potential of -50 mV, tamoxifen produced significantly greater inhibition of I ⁇ r compared to quinidine (84.8% ⁇ 1.3% versus 42.5% ⁇ 9.1%, p ⁇ .01). Thus, tamoxifen was amore potent and longer lasting blocker of I ⁇ ⁇ than quinidine.
• FIG. 6 shows action potentials recorded before and after 4 min. exposure of 3.3 ⁇ mol/L tamoxifen. Su ⁇ risingly, although tamoxifen inhibited I ⁇ r by about 84.8% at this concentration, no significant prolongation of APD was observed.
• APD measured at 90% repolarization (APD 90 ) before and after about 4-5 min. superfusion of tamoxifen (3.3 ⁇ mol/L) was 341 ⁇ 49 ms and 332 ⁇ 19 ms respectively (n 16, p>0.05). Since under control conditions, no significant shortening of APD was observed in the initial 10 min. after cell membrane rapture, the absence of APD prolongation by tamoxifen was not secondary to a "rundown" phenomenon.
• Example 4 ECG Arrhythmia Assessment of Ibutilide Highly accurate and reproducible measures of the QT interval in humans have been developed (Woosley et al, Am. J. Cardiol 72: 36B-43B (1993); Sale et al, Clin. Pharmacol Ther. 56: 295-301 (1994)).
• ECG analysis is one method of assessing arrhytl mias, such as prolonged QT intervals.
• Ibutilide or any other pharmaceutical agent to be studied can be assessed using an ECG. It is preferable to administer the drag (e.g., ibutilide) intravenously, ibutilide produces a reliable prolongation of the QT interval, and its effects dissipate rapidly.
• ibutilide 0.003 mg/kg
• ECGs were coded, randomized and blindly measured using a computer- operator interactive program, employing a validated method as previously described in ((Woosley et al, (1993); Sale et al, (1994)). Women were studied at each phase of the menstraal cycle (e.g., menses, ovulation and luteal), guided by luteinising hormone (LH) surge and confirmed with estradiol and progesterone plasma determinations. Men were studied only once. The maximum and average QT changes, after each dose of ibutilide, were compared to baseline.
• LH luteinising hormone
• Example 5 Role of delayed rectifier potassium current in spontaneously beating cardiomyocytes Delayed rectifier potassium channels are important components of cardiac repolarization. There are two major delayed rectifier potassium currents, I ⁇ r (rapid component) and I KS (slow component). Drags that block these currents, particularly I ⁇ r, slow cardiac repolarization and increase the risk of developing potentially fatal cardiac arrhythmias such as Torsades de Pointes. Moreover, mutations in the genes encoding for delayed rectifier potassium channel proteins have been linked to long QT syndrome, a condition seen in patients at high risk for developing Torsades de Pointes cardiac arrhythmias and sudden death.
• I ⁇ r rapid component
• I KS slow component
• I ⁇ r While several specific inhibitors of I ⁇ r (d-sotalol, dofetilide, E-4031) have been available for several years, studies of endogenous I KS have been hampered by a lack of pharmacological tools to selectively block its activity. Recently, however, a new compound, chromanol 293B, has been reported to be a relatively selective blocker of I KS (Busch et al, Pflug. Arch. 432: 1094-6 (1996)). hi the present report, we have tested this compound using spontaneously beating cultures of neonatal rat cardiomyocytes.
• chromanol 293B caused a dose-dependent decrease in beating rate, but even at the highest drag concentration tested (100 mM), the reduction in beating rate was only about 50% that of control, hi guinea pig ventricular myocytes, the IC5 0 value for 293B inhibition of I KS is approximately 2 mM, although concentrations as high as 100 mM were required to achieve complete blockade of I KS in those cells (Busch et al, (1996)).

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